All lanes : Anti-Calnexin antibody [EPR3632] (ab92573) at 1/20000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : CANX knockout HEK-293T cell lysate
Lane 3 : U-2 OS cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab92573).

Lanes 1 - 4: Merged signal (red and green). Green - ab92573 observed at 80 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab92573 was shown to react with Calnexin in wild-type HEK-293T cells in Western blot with loss of signal observed in CANX knockout cell line ab255368 (CANX knockout cell lysate ab263805). Wild-type HEK-293T and CANX knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab92573 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 20000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

ab92573 staining Calnexin in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab92573 at 1/1000 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92573).

All lanes : Anti-Calnexin antibody [EPR3632] (ab92573) at 1/20000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : Calnexin knockout HAP1 cell lysate
Lane 3 : THP-1 cell lysate
Lane 4 : RAW 264.7 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 68 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab92573 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab92573 was shown to specifically react with Calnexin when Calnexin knockout samples were used. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab92573 and ab8245 (loading control to GAPDH) were diluted at 1/20,000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92573).

Immunocytochemistry/Immunofluorescence analysis of human lung carcinoma A549 cells labelling Calnexin with ab92573. Cells were fixed with 4% PFA in PBS for 15 minutes washed three times with PBS, and then washed twice with 25 mM glycine–PBS. The cells were treated with cold methanol and washed three times with 0.3% Triton X-100 in PBS for permeabilization. The fixed cells were blocked with 5% BSA in PBS overnight. An Alexa Fluor^® 594-conjugated anti-rabbit was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92573).

Immunohistochemical analysis of paraffin embedded Human tonsil tissue using ab92573 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92573).